This grant proposes collaborative studies between investigators at Baylor College of Medicine (BCM) and The University of Rochester Medical School to develop new diagnostic assays for the Norwalk group of viruses. Norwalk virus is one of the most important viral pathogens causing acute epidemic gastroenteritis. Epidemiology studies indicate that transmission of Norwalk virus is through water-borne, food-borne or person-to-person routes. Norwalk virus is a 27-nm virus and virus particles reportedly contain a single structural polypeptide with an apparent molecular weight of 59,000. The morphology of some virions and the presence of a single structural protein are features similar to viruses in the Caliciviridae family. Based on recent studies with cDNAs made in the last year at BCM, the nucleic acid of Norwalk virus has been shown to be single-stranded RNA. We will use molecular approaches to completely characterize the Norwalk virus genome and develop new diagnostic assays for Norwalk virus. Characterization of the Norwalk virus genome will be done using specific cloned DNA made at BCM in libraries of pUC-13 or in the lambda ZAPII vector. Specific cDNAs identified by nucleic acid hybridization (and new ones to be identified) will be characterized until cDNA clones representing the entire genome are found. These clones will be used for nucleotide sequencing to determine the genome organization of this virus. Furthermore, cDNA clones encoding immunoreactive proteins will be identified, subcloned and expressed using a baculovirus expression system. Each expressed protein will be used as an immunogen to produce immunologic reagents. Methods of virus detection based on diagnostic assays using prepared hyperimmune antiserum and monoclonal antibodies or based on detection of nucleic acid by polymerase chain reaction (PCR) will be developed and compared. We aim to determine what assay(s) will be (1) the most rapid, sensitive and specific for detection of Norwalk virus and (2) capable of detecting Norwalk virus only or Norwalk virus and Norwalk-like viruses [Snow Mountain agent (SMA), Hawaii agent] and possibly human caliciviruses.